Table 2. Primers used to construct B19 VP1u point mutations and truncated VP1u.

Name		Sequence (5′–3′)
Point mutations
Group I (Catalytic network)
VP1u(Y130A)	Forward	CCCGGTACTAACGCTGTTGGGCCTGGC
	Reverse	GCCAGGCCCAACAGCGTTAGTACCGGG
	Forward	ACTAACTATGTTGCGCCTGGCAATGAG
	Reverse	CTCATTGCCAGGCGCAACATAGTTAGT
	Forward	GCAAGGATTCATGACTTTAGGTATAGC
	Reverse	GCTATACCTAAAGTCATGAATCCTTGC
Group II (Binding calcium ions)
VP1u(H153A)	Forward	GCTGCAAGGATTGCTGACTTTAGGTAT
	Reverse	ATACCTAAAGTCAGCAATCCTTGCAGC
VP1u(Y157F)	Forward	ATGACTTTAGGTTTAGCCAACTGGCT
	Reverse	AGCCAGTTGGCTAAACCTAAAGTCATG
VP1u(Y168F)	Forward	GGAATAAATCCATTTACTCATTGGACT
	Reverse	AGTCCAATGAGTAAATGGATTTATTCC
VP1u(D175A)	Forward	TGGACTGTAGCAGCTGAAGAGCTTTTA
	Reverse	TAAAAGCTCTTCAGCTGCTACAGTCCA
VP1u(D195A)	Forward	CAAGTAGTAAAAGCCTACTTTACTTTA
	Reverse	TAAAGTAAAGTAGGCTTTTACTACTTG
Group III (Phospholipid environment)
VP1u(K162R)	Forward	AGCCAACTGGCTAGGTTGGGAATAAATCCA
	Reverse	TGGATTTATTCCCAACCTAGCCAGTTGGCT
Group IV (No-conserved)
VP1u(P133R)	Forward	AACTATGTTGGGCGTGGCAATGAGCTA
	Reverse	TAGCTCATTGCCACGCCCAACATAGTT
VP1u(A207Y)	Forward	GCTGCCCCTGTGTACCATTTTCAAGGA
	Reverse	TCCTTGAAAATGGTACACAGGGGCAGC
Wild type and truncated VP1u
VP1u(WT)	Forward	CGGGATCCATGAGTAAAGAAAGTGGCAAATG
	Reverse	CCCAAGCTTCCTGCAGAATTAACTGAAGTCATGCT
VP1u(ΔN11)	Forward	CGGGATCCGATGATGAATTTGCTAAAGCTGT
	Reverse	CCCAAGCTTCCTGCAGAATTAACTGAAGTCATGCT
VP1u(ΔN21)	Forward	CGGGATCCCAATTTGTGGAATTTTATGAAAA
	Reverse	CCCAAGCTTCCTGCAGAATTAACTGAAGTCATGCT
VP1u(ΔN42)	Forward	CGGGATCCGATCATTATAATATTTCTTTAGA
	Reverse	CCCAAGCTTCCTGCAGAATTAACTGAAGTCATGCT
VP1u(ΔC6)	Forward	CGGGATCCATGAGTAAAGAAAGTGGCAAATG
	Reverse	CCCAAGCTTCGGCGTTGTAAGCGGGAACTTCCG
VP1u(ΔC15)	Forward	CGGGATCCATGAGTAAAGAAAGTGGCAAATG
	Reverse	CCCAAGCTTCACTTCCTTGAAAATGGGCCACAG
VP1u(ΔC23)	Forward	CGGGATCCATGAGTAA AGAAAGTGGCAAATG
	Reverse	CCCAAGCTTCGGCAGCTGCACCTTTTAAAGTAT

^aAll sequences are based on the sequence of the J35 isolate (GenBank accession no. AY386330).

^b GGATCC is a restriction site of BamHI, AAGCTT is a restriction site of HindIII.